Summary The microtubule (MT) cytoskeleton and the molecular motors that move along it-dynein and kinesin-are responsible for powering the movement of chromosomes during mitosis and of organelles, signaling molecules and RNAs in the cytoplasm. The spatial and temporal regulation involved in transporting these cargos at the cellular level remains one of the big unsolved questions in the field of cell biology. I propose to use the filamentous fungus, Aspergillus nidulans, as a model system to dissect the molecular mechanisms of MT-based transport, with a combination of approaches ranging from genome-wide screens to single- molecule biophysics. Aspergillus polarized hyphae, whose rapid growth requires MT-based transport, and its high frequency of homologous recombination make it an ideal model organism for studying transport. Importantly, the number and types of cargo transporting motors present in Aspergillus are more similar to mammalian systems than to yeast-like fungi. We will identify all the organelles transported by the Aspergillus motors; this will constitute the first inventory of cargos carried by MT-based motors in a single cell. After identifying these cargos, we will create a complete gene disruption library that will be used to perform high-throughput microscopy-based screens to identify novel molecules required for dynein- or kinesin-based motility. In parallel with screening, we will purify the native Aspergillus motors and determine their properties in vitro using single molecule motility assays. Hits from our screens that pass secondary rounds of screening will be tested in these assays for roles in regulating motor function or cargo binding. Ultimately, we aim to reconstitute motor-cargo transport in vitro and to develop methods to observe the dynamics of transport in vivo with nanometer precision. We expect to identify novel conserved paradigms regarding the mechanism of MT-based cargo transport.